Immuno-FISH - Revision history

Admin at 13:48, 2 March 2022

2022-03-02T13:48:20Z

← Older revision		Revision as of 13:48, 2 March 2022
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	**If using a [https://www.jenabioscience.com/probes-epigenetics/dna-cdna-labeling/fluorescent-dna-cdna-labeling/fluorescent-nick-translation-labeling-kits/pp-305-647n-atto647n-nt-labeling-kit Jenna Kit]:		**If using a [https://www.jenabioscience.com/probes-epigenetics/dna-cdna-labeling/fluorescent-dna-cdna-labeling/fluorescent-nick-translation-labeling-kits/pp-305-647n-atto647n-nt-labeling-kit Jenna Kit]:
	**#Process 1.5 μg of DNA & collect using a monarch kit into 50 μl		**#Process 1.5 μg of DNA & collect using a monarch kit into 50 μl
	**#Use a 1:2500 dilution of the ~~labeled~~ probes for staining		**#Use a 1:2500 dilution of the labelled probes for staining
	* Antibody(s) targeting proteins of interest.		* Antibody(s) targeting proteins of interest.
	* Secondary Fab's if the antibody is not directly labelled.		* Secondary Fab's if the antibody is not directly labelled.
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	== Immunostaining ==		== Immunostaining ==

	# ~~Grow and stimulate cells~~ as ~~required on #1~~.~~5 thickness coverslips~~.		# Immunostain as per [https://wiki.phagocytes.ca/index.php/Immunostaining our standard intracellular procedure].
	~~# Fix with 4% PFA in PBS, 15 min, 37C~~.		#After labelling and washing perform a light secondary fixation, 2% PFA in PBS, 10 min, room temperature.
	~~# Rinse 2X with PBS~~.
	# ~~Wash 1 x 10 min with antibody buffer.~~
	~~# Wash 1 x 10 min with antibody high-wash buffer.~~
	~~# Wash 1 x 10 min with antibody buffer.~~
	~~# Add primary antibody to coverslip at the desired dilution in antibody buffer; incubate 1 hr at room temperature~~, ~~or overnight at 4C.~~
	~~# Wash 3 x 10 min with antibody low-wash buffer.~~
	~~# Repeat steps 4-8 with secondary antibody (if needed).~~
	~~#Lightly fix in~~ 2% PFA in ~~pBS~~, 10 min, room temperature.
	#Rinse 2x with PBS.		#Rinse 2x with PBS.
	# It may be a good idea to confirm antibody labelling before moving onto FISH staining.		# It may be a good idea to confirm antibody labelling before moving onto FISH staining.
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	# Dilute the FISH probe in hybridization solution, and heat at 75C for 10 min.		# Dilute the FISH probe in hybridization solution, and heat at 75C for 10 min.
	# Incubate denatured probe at 37C for 10 min. During this time denature your coverslip.		# Incubate denatured probe at 37C for 10 min. During this time denature your coverslip (steps 3-4, below).
	# Denature the coverslip in denaturation solution, 70C, 2 min		# Denature the coverslip in denaturation solution, 70C, 2 min
	# Dehydrate the slide by immersing the coverslip in 75%, 90% then 100% ethanol, 2 min per immersion. Air dry slide.		# Dehydrate the slide by immersing the coverslip in 75%, 90% then 100% ethanol, 2 min per immersion. Air dry slide.

Admin at 12:43, 2 March 2022

2022-03-02T12:43:07Z

← Older revision		Revision as of 12:43, 2 March 2022
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	* Purified FISH probes, ~25 uM. Typically, a small plasmid (pluescript) bearing 2-3 kb of the target region of the genome, nick-labelled with the desired fluorphore and digested into 200-500 bp fragments.		* Purified FISH probes, ~25 uM. Typically, a small plasmid (pluescript) bearing 2-3 kb of the target region of the genome, nick-labelled with the desired fluorphore and digested into 200-500 bp fragments.
			**If using a [https://www.jenabioscience.com/probes-epigenetics/dna-cdna-labeling/fluorescent-dna-cdna-labeling/fluorescent-nick-translation-labeling-kits/pp-305-647n-atto647n-nt-labeling-kit Jenna Kit]:
			**#Process 1.5 μg of DNA & collect using a monarch kit into 50 μl
			**#Use a 1:2500 dilution of the labeled probes for staining
	* Antibody(s) targeting proteins of interest.		* Antibody(s) targeting proteins of interest.
	* Secondary Fab's if the antibody is not directly labelled.		* Secondary Fab's if the antibody is not directly labelled.

Admin: /* General: */

2022-01-26T13:19:35Z

General:

← Older revision		Revision as of 13:19, 26 January 2022
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	=== Denaturation Solutions ===		=== Denaturation Solutions ===

	* 20X SSC stock: 3M NaCl, 300 mM sodium citrate		* 20X SSC stock: 3M NaCl, 300 mM sodium citrate, pH 7.0
	* Denatuation solution: 70% deionized foramide in 2X SSC		* Denatuation solution: 70% deionized foramide in 2X SSC
	* Hybridization solution: 50% deionized foramide, 10% dextran sulfate in 2X SSC		* Hybridization solution: 50% deionized foramide, 10% dextran sulfate in 2X SSC

Admin: /* General: */

2022-01-20T18:28:40Z

General:

← Older revision		Revision as of 18:28, 20 January 2022
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	=== General: ===		=== General: ===

	* Purified FISH probes. Typically, a small plasmid (pluescript) bearing 2-3 kb of the target region of the genome, nick-labelled with the desired fluorphore and digested into 200-500 bp fragments.		* Purified FISH probes, ~25 uM. Typically, a small plasmid (pluescript) bearing 2-3 kb of the target region of the genome, nick-labelled with the desired fluorphore and digested into 200-500 bp fragments.
	* Antibody(s) targeting proteins of interest.		* Antibody(s) targeting proteins of interest.
	* Secondary Fab's if the antibody is not directly labelled.		* Secondary Fab's if the antibody is not directly labelled.

Admin at 16:42, 11 January 2022

2022-01-11T16:42:40Z

← Older revision		Revision as of 16:42, 11 January 2022
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	This is a protocol for performing Fluorescence In-Situ Hybridization alongside immunostained cells. The protocol assumes that the cells are already grown on a coverslip, treated, and are ready for imaging. Note that this protocol uses an ethanol dehydration/denaturation step, and as such is unlikely to work with fluorescent proteins. This protocol also assumes that you have directly labelled (e.g. fluorescent) FISH probes. Protocol is modified from Ye ''at al'' (2009)<ref>C. J. Ye, L. Lawrenson, G. Liu, J. B. Stevens, K. J. Ye, S. W. Bremer, and H. H. Heng, Chapter 19: Simultaneous fluorescence immunostaining and FISH. In Liehr edited: Fluorescence in situ hybridization (FISH) - Application. Springer Publishing. 193-216 (2009). https://link.springer.com/protocol/10.1007/978-3-540-70581-9_19</ref>.		'''Note: this is a protocol in development.''' This is a protocol for performing Fluorescence In-Situ Hybridization alongside immunostained cells. The protocol assumes that the cells are already grown on a coverslip, treated, and are ready for imaging. Note that this protocol uses an ethanol dehydration/denaturation step, and as such is unlikely to work with fluorescent proteins. This protocol also assumes that you have directly labelled (e.g. fluorescent) FISH probes. Protocol is modified from Ye ''at al'' (2009)<ref>C. J. Ye, L. Lawrenson, G. Liu, J. B. Stevens, K. J. Ye, S. W. Bremer, and H. H. Heng, Chapter 19: Simultaneous fluorescence immunostaining and FISH. In Liehr edited: Fluorescence in situ hybridization (FISH) - Application. Springer Publishing. 193-216 (2009). https://link.springer.com/protocol/10.1007/978-3-540-70581-9_19</ref>.

	== Reagents ==		== Reagents ==

Admin: /* General: */

2021-11-18T19:09:19Z

General:

← Older revision		Revision as of 19:09, 18 November 2021
Line 9:		Line 9:
	* Secondary Fab's if the antibody is not directly labelled.		* Secondary Fab's if the antibody is not directly labelled.
	* 2% and 4% PFA in PBS		* 2% and 4% PFA in PBS
	* 0.01% sodium azide in PBS
	* 1% sodium azide in water
	* Antibody buffer: 3% BSA in PBS		* Antibody buffer: 3% BSA in PBS
	* Antibody buffer with 1% Triton X-100		* Antibody high wash: antibody buffer with 1% Triton X-100
	* Antibody wash ~~buffer~~: ~~antibody~~ buffer diluted 1:10 with PBS ~~+ 0.1% Triton X-100~~		* Antibody low wash: high-wash buffer diluted 1:10 with PBS

	=== Denaturation Solutions ===		=== Denaturation Solutions ===
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	# Rinse 2X with PBS.		# Rinse 2X with PBS.
	# Wash 1 x 10 min with antibody buffer.		# Wash 1 x 10 min with antibody buffer.
	# Wash 1 x 10 min with antibody buffer ~~+ 1% Triton X-100~~.		# Wash 1 x 10 min with antibody high-wash buffer.
	# Wash 1 x 10 min with antibody buffer.		# Wash 1 x 10 min with antibody buffer.
	# Add primary antibody to coverslip at the desired dilution in antibody buffer; incubate 1 hr at room temperature, or overnight at 4C.		# Add primary antibody to coverslip at the desired dilution in antibody buffer; incubate 1 hr at room temperature, or overnight at 4C.
	# Wash 3 x 10 min with antibody wash buffer.		# Wash 3 x 10 min with antibody low-wash buffer.
	# Repeat steps 4-8 with secondary antibody (if needed).		# Repeat steps 4-8 with secondary antibody (if needed).
	#Lightly fix in 2% PFA in pBS, 10 min, room temperature.		#Lightly fix in 2% PFA in pBS, 10 min, room temperature.

Admin at 19:07, 18 November 2021

2021-11-18T19:07:36Z

@@ Line 1: / Line 1: @@
-This is a protocol for performing Fluorescence In-Situ Hybridization alongside immunostained cells. The protocol assumes that the cells are already grown on a coverslip, treated, and are ready for imaging. Note that this protocol uses an ethanol dehydration/denaturation step, and as such is unlikely to work with fluorescent proteins. This protocol also assumes that you have directly labelled (e.g. fluorescent) FISH probes.
+This is a protocol for performing Fluorescence In-Situ Hybridization alongside immunostained cells. The protocol assumes that the cells are already grown on a coverslip, treated, and are ready for imaging. Note that this protocol uses an ethanol dehydration/denaturation step, and as such is unlikely to work with fluorescent proteins. This protocol also assumes that you have directly labelled (e.g. fluorescent) FISH probes. Protocol is modified from Ye ''at al'' (2009)<ref>C. J. Ye, L. Lawrenson, G. Liu, J. B. Stevens, K. J. Ye, S. W. Bremer, and H. H. Heng, Chapter 19: Simultaneous fluorescence immunostaining and FISH.  In Liehr edited: Fluorescence in situ hybridization (FISH) - Application.  Springer Publishing.  193-216 (2009). https://link.springer.com/protocol/10.1007/978-3-540-70581-9_19</ref>.
 == Reagents ==
@@ Line 19: / Line 19: @@
 * 20X SSC stock: 3M NaCl, 300 mM sodium citrate
 * Denatuation solution: 70% deionized foramide in 2X SSC
 * Hybridization solution: 50% deionized foramide, 10% dextran sulfate in 2X SSC
 * Hybridization wash: 50% deionized foramide in 2X SSC
 == Immunostaining ==
@@ Line 32: / Line 31: @@
 # Wash 1 x 10 min with antibody buffer + 1% Triton X-100.
 # Wash 1 x 10 min with antibody buffer.
-# Add primary antibody to slide at the desired dilution in antibody buffer; incubate 1 hr at room temperature, or overnight at 4C.
+# Add primary antibody to coverslip at the desired dilution in antibody buffer; incubate 1 hr at room temperature, or overnight at 4C.
 # Wash 3 x 10 min with antibody wash buffer.
 # Repeat steps 4-8 with secondary antibody (if needed).
 # It may be a good idea to confirm antibody labelling before moving onto FISH staining.

Admin: Created page with "This is a protocol for performing Fluorescence In-Situ Hybridization alongside immunostained cells. The protocol assumes that the cells are already grown on a coverslip, treat..."

2021-11-18T18:50:15Z

Created page with "This is a protocol for performing Fluorescence In-Situ Hybridization alongside immunostained cells. The protocol assumes that the cells are already grown on a coverslip, treat..."

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This is a protocol for performing Fluorescence In-Situ Hybridization alongside immunostained cells. The protocol assumes that the cells are already grown on a coverslip, treated, and are ready for imaging. Note that this protocol uses an ethanol dehydration/denaturation step, and as such is unlikely to work with fluorescent proteins. This protocol also assumes that you have directly labelled (e.g. fluorescent) FISH probes.

== Reagents ==

=== General: ===

* Purified FISH probes. Typically, a small plasmid (pluescript) bearing 2-3 kb of the target region of the genome, nick-labelled with the desired fluorphore and digested into 200-500 bp fragments.
* Antibody(s) targeting proteins of interest.
* Secondary Fab's if the antibody is not directly labelled.
* 2% and 4% PFA in PBS
* 0.01% sodium azide in PBS
* 1% sodium azide in water
* Antibody buffer: 3% BSA in PBS
* Antibody buffer with 1% Triton X-100
* Antibody wash buffer: antibody buffer diluted 1:10 with PBS + 0.1% Triton X-100

=== Denaturation Solutions ===

* 20X SSC stock: 3M NaCl, 300 mM sodium citrate
* Denatuation solution: 70% deionized foramide in 2X SSC
* Detection solution: 1% BSA + 0.05% TWEEN 20 in 4X SSC
* Detection wash: 0.05% TWEEN 20 in 4X SSC
* Hybridization solution: 50% deionized foramide, 10% dextran sulfate in 2X SSC
* Hybridization wash: 50% deionized foramide in 2X SSC

== Immunostaining ==

# Grow and stimulate cells as required on #1.5 thickness coverslips.
# Fix with 4% PFA in PBS, 15 min, 37C.
# Rinse 2X with PBS.
# Wash 1 x 10 min with antibody buffer.
# Wash 1 x 10 min with antibody buffer + 1% Triton X-100.
# Wash 1 x 10 min with antibody buffer.
# Add primary antibody to slide at the desired dilution in antibody buffer; incubate 1 hr at room temperature, or overnight at 4C.
# Wash 3 x 10 min with antibody wash buffer.
# Repeat steps 4-8 with secondary antibody (if needed).
# It may be a good idea to confirm antibody labelling before moving onto FISH staining.